A major goal of this lab is to study the structure and function of Sendai virus proteins as they relate to viral pathogenicity. Sendai virus is a murine parainfluenza virus similar in structure to the influenza virus, a virus that continues to be a major cause of human injection in modern man. Knowledge gained from studying Sendai virus may be applied to influenza. Sendai virus is pneumotropic in mice while FI-R, a variant, as pantropic and causes a systemic infection in mice. Enhance proteolytic cleavability of the F protein, due to an F mutation adjacent to the cleavage site, and bipolar budding of the virus, resulting from a disruption of the pantropic determinants. More mutant viruses with enhance cleavability and bipolar budding phenotypes have been isolated. The M and F genes of these viruses contain mutations different from those found in the M and F genes of F1-R. These viruses do not disrupt the microtubule network of the cells and they are incapable of causing a systemic injection in mice. Thus, a disruption of the microtubule network, a phenotype unique to F1-R, correlates, better than bipolar budding, with the pantropism of the virus. The results suggest that the specific mutation adjacent to the cleavage site of F and the two mutations in M confer upon F1-R the ability to cause a systemic infection. The first specific aim of this project is to prove this by using reverse genetics to create a recombinant virus containing only those mutations. The tropism of the recombinant virus will be determined. The second specific aim is to investigate the molecular nature of the microtubule disruption caused by F1-R M. Preliminary results from a West-western analysis suggest a direct interaction between M and tubulin. The analysis will be repeated with wild-type and F1 R M under varying conditions to assess the molecular nature of the interaction. In addition, the relative level of total tubulin in F1-R M expressing ells and the ability of taxol to counteract the microtuble depolymerization will be determined. A third specific aim is to determine the gene or mutation responsible for the bipolar budding of the mutant viruses that fail to disrupt the microtubule network of MDCK cells. The ability of the mutant M proteins to alter the apical transport of wild-type F will be assessed suing a surface biotinylation assay. The HN genes will be sequenced and the polarized transport of individual expressed F and HN proteins will be determined.